Poly(amidoamine) Dendrimer-Coordinated Copper(II) Complexes as a

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PAMAM Dendrimer-Coordinated Copper(II) Complexes as a Theranostic Nanoplatform for Radiotherapy-Enhanced MR Imaging and Chemotherapy of Tumors and Tumor Metastasis Yu Fan, Jiulong Zhang, Menghan Shi, Dan Li, Chunhua Lu, Xueyan Cao, Chen Peng, Serge Mignani, Jean Pierre Majoral, and Xiangyang Shi Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b04757 • Publication Date (Web): 30 Jan 2019 Downloaded from http://pubs.acs.org on January 31, 2019

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PAMAM Dendrimer-Coordinated Copper(II) Complexes as a Theranostic Nanoplatform for Radiotherapy-Enhanced MR Imaging and Chemotherapy of Tumors and Tumor Metastasis Yu Fan1, Jiulong Zhang2, Menghan Shi1, Dan Li1, Chunhua Lu3, Xueyan Cao1, Chen Peng2,4*, Serge Mignani5*, Jean-Pierre Majoral6,7*, Xiangyang Shi1,5*

1

State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of

Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, People’s Republic of China. E-mail: [email protected]; Phone: +86-21-67792656 2

Department of Radiology, Shanghai Public Health Clinical Center, Fudan University, Shanghai

201508, People’s Republic of China 3

Department of Radiotherapy, Shanghai Tenth People’s Hospital, Tongji University School of

Medicine, Shanghai 200072, People’s Republic of China 4

Cancer Center, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai

200072, People’s Republic of China. E-mail: [email protected]; Phone: +86-21-66301477 5

CQM - Centro de Química da Madeira, MMRG, Universidade da Madeira, Campus da Penteada,

9020-105 Funchal, Portugal. E-mail: [email protected]; Phone: +33-688069293 6

Laboratoire de Chimie de Coordination du CNRS, 205 route de Narbonne, 31077 Toulouse Cedex 4,

France. E-mail: [email protected], Phone: +33-561333123 7

Université de Toulouse, UPS, INPT, F-31077 Toulouse Cedex4, France

______________________ * To whom correspondence should be addressed. 1 ACS Paragon Plus Environment

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ABSTRACT Development of a powerful nanoplatform to realize simultaneous therapy and diagnosis of cancer using similar element for theranostics remains a critical challenge. Herein, we report such a theranostic nanoplatform based on pyridine (Pyr)-functionalized generation 5 (G5) poly(amidoamine) (PAMAM) dendrimers complexed with copper(II) (Cu(II)) for radiotherapy-enhanced T1-weighted magnetic resonance (MR) imaging and synergistic radio-chemotherapy of both tumors and tumor metastasis. In this study, amine-terminated G5 dendrimers were covalently linked with 2-pyridinecarboxylic acid, acetylated to neutralize their remaining terminal amines, and complexed with Cu(II) through both the internal tertiary amines and surface Pyr groups to form the G5.NHAc-Pyr/Cu(II) complexes. We show that the complexes are able to inhibit the proliferation of different cancer cell lines with half maximal inhibitory concentrations (IC50s) ranging from 4 to 10 µM and induce significant cancer cell apoptosis. Due to the presence of Cu(II), the G5.NHAc-Pyr/Cu(II) complexes display an r1 relaxivity of 0.7024 mM−1s−1, enabling effective in vivo MR imaging of tumor xenografts and lung metastatic nodules. Further, under a radiotherapy (RT) condition, the tumor MR imaging sensitivity can be significantly enhanced, and the G5.NHAc-Pyr/Cu(II) complexes enable enhanced chemotherapy of both a xenografted tumor model and a blood vessel metastasis model. With the truly demonstrated theranostic potential of the dendrimer-Cu(II) nanocomplexes without additional agents or elements for RTenhanced MR imaging and chemotherapy of tumor and tumor metastasis, this novel Cu(II)-based nanohybrids may hold a great promise for theranostics of different cancer types and metastases.

KEYWORDS:

dendrimers-Cu(II)

complexes;

oxidative

stress;

radiotherapy;

chemotherapy;

nanotheranostics

2 ACS Paragon Plus Environment

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Nanotechnology, by opening the world beyond microscale, is recognized as one of the major “Key Enabling Technologies” in the progress of biology fields, particularly the medical sciences.1-3 Over recent decades, the development of theranostic nanomedicine platforms (nanotheranostics), which simultaneously integrate diagnosis and therapy elements using various nanocarriers, has been one of the hot spots in the nanomedicine field.4 Indeed, the rapidly growing theranostics provides a very promising strategy to achieve enhanced and efficient therapy, real-time monitoring, and comprehensive understanding of cancer treatment in personalized medicine with reduced treatment time and cost.5 In general, a traditional nanotheranostic agent is the combination of therapy and imaging agents in a single nanoplatform. These essential building blocks, including nanoplatform, therapeutic agent and imaging agent, have been under extensive investigation to design and build these efficient and smart nanotheranostics, in which each functional segment works synergistically and efficiently. Rather, it will require the careful coordination of diagnosis and therapy, longer process during the manufacturing of these theranostic agents, and much more characterization compared to simple drug molecules or diagnostic agents. Presently, most multifunctional theranostic systems are still in the proof of concept stage, for which the imaging and therapy are performed essentially independently, rather than in an integrated protocol.6 In this sense, there are still a lot of challenges to overcome regarding the translation of theranostic nanomedicine from basic research into clinic.7 An important consideration in the development of new theranostics is the appropriate nanoplatform integrating the therapeutic and imaging agents in one entity, thus decreasing the gap between diagnosis and treatment with minimized separate comprehensions. Over the past few decades, many theranostic nanoplatforms have been developed such as carbon nanotubes, liposomes, albumin-based particles, biodegradable polymer composites, polymeric micelles, dopamine-based nanomaterials, inorganic particles, dendrimers and dendrons,8-14 whereas dendrimers and dendrons can also be used as drug per se.15-17 Based on the precise multistep synthesis, multiple surface chemical functionality, and the 3 ACS Paragon Plus Environment

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monodispersity of dendrimer, dendrimers are ideal nanoplatforms to act as hosts to incorporate or conjugate a range of diverse types of molecules or particles for tumor theranostics. For example, poly(amidoamine) (PAMAM) dendrimers have been developed as versatile nanoplatforms to deliver drugs, genes and diagnostic agents via various administration routes including intravenous, oral, nasal, intramuscular injection, etc. for better cancer imaging and/or therapy.18,

19

A wide range of literature

about dendrimers is available today.20-23 Strongly, these positive results encouraged us to develop original dendrimer-based platforms in the nanotheranostic domain. Since the discovery of cisplatin and derivatives, numerous efforts have been undertaken to find and develop new metal-based complexes including metallodendrimers, which represent a powerful strategy to provide new nanomedicine in the oncology field.24 It has been highlighted that copper(II) (Cu(II))conjugated dendrimers were synthesized and screened for antiproliferative activity against several human cancer cell lines. For instance, Zhao and co-workers used G0 PAMAM dendrimers to construct heptanuclear Cu(II) metallodendrimers bearing four Schiff base moieties on the periphery to coordinate Cu(II). And the other three Cu(II) ions are complexed with amide groups and the nitrogen sites within the dendrimer void spaces.25 This multi-nuclear Cu(II)-based dendrimer complexes displayed moderate in vitro cytotoxicity against MOLT-4 leukemia and MCF-7 breast cancer cell lines (IC50s are around 10 µM), which is close to those of cisplatin. Recently, we highlighted the synthesis of original G1-G3 Cu(II)complexed phosphorus dendrimer grafted with N-(pyridin-2-ylmethylene) ethanamine groups on the surface (12, 24, and 48 chelating groups, respectively). The third generation Cu(II)-complexed phosphorus dendrimer (1G3-Cu) was shown to be the most potent derivative displaying good anticancer properties against a number of cancerous cell lines, with an IC50s range of 0.3-1.6 µM.26 Importantly, the Cu(II) complexes were found more cytotoxic toward cancerous cell lines versus noncancer cell lines, having a good safety ratio.27 Biochemical studies showed that the free dendrimers and the corresponding Cu(II) complexes behaved differently inside the cells. The free dendrimers activated 4 ACS Paragon Plus Environment

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Caspase-3, whereas the complexes strikingly reduced the Caspase-3 activity, and induced the translocation of Bax to the mitochondria, resulting in severe DNA fragmentation.27, 28 Importantly, Cu(II) has also been regarded as an alternative T1-weighted MR contrast agent owing to the presence of unpaired electrons in its outermost orbital.29 Previous work showed that the Cu2−xS nanoparticles (NPs) (0.26 mM−1s−1),30 the CuO NPs (0.38 mM−1s−1),31 and Cu nanoclusters (0.35 mM−1s−1)32 were able to be used as contrast agents for in vitro or in vivo MR imaging applications. Moreover, it was reported that Cu(II)-based oleate nanocolloids possessed a relatively large r1 relaxivity (4.26 mM−1s−1),33 quite similar to commercial Magnevist (4.56 mM−1s−1).34 Although Cu(II)-based agents can be used for both therapeutic and imaging purposes, no literature reports have addressed the use of them for simultaneous tumor therapy and diagnosis. Currently, the combination strategy of the available cancer therapeutic approaches, such as chemo/photothermal,35 photodynamic/photothermal,36, 37 or radio/photothermal therapies,37 has usually led to unprecedented synergistic therapeutic efficacy.38, 39 It is increasingly evident that the radiotherapy (RT)-induced changes in tumor vasculature and interstitial fluid pressure can modulate the NP accumulation and their spatial localization in tumors.40,

41

Hence, combining RT with nanomedicine

represents a novel approach to overcoming the tumor microenvironmental barriers and improving treatment efficacy.42-44 The aim of this current study is to design and explore the Cu(II)-based nanotheranostics to fully expand the current view of cancer theranostics, which greatly simplify the design of the theranostic systems. Here, we report the minimalist design of original PAMAM dendrimer-coordinated Cu(II) complexes to construct a theranostic nanoplatform for MR imaging and combination RT/chemotherapy against tumors and tumor metastasis (Figure 1A). In addition, the RT-induced enhancement of MR imaging of tumors was also explored. In our work, firstly, amine-terminated G5 PAMAM dendrimers were covalently conjugated with 2-pyridinecarboxylic acid (Pyr-COOH) and the remaining amines of 5 ACS Paragon Plus Environment

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dendrimers were acetylated, followed by complexation of Cu(II) via both ligation of Pyr and dendrimer tertiary amines. The therapeutic activity of G5.NHAc-Pyr/Cu(II) complexes were explored by cytotoxicity, apoptosis, oxidative stress detection, cell cycle and western blot assays. Next, we performed the MR phantom studies of these complexes and used them for RT-enhanced T1-weighted MR imaging and chemotherapy using the xenografted mouse breast cancer model. And we also explored the use of these complexes for MR imaging and combination RT/chemotherapy of blood vessel metastasis model. To the best of our knowledge, this is the first report related to PAMAM dendrimer-coordinated Cu(II) complexes for nanotheranostics of tumors and tumor metastasis.

Figure 1. (A) Schematic diagram illustrating the synthesis of Cu(II) complexes with Pyr-functionalized PAMAM dendrimers for RT-enhanced T1 MR imaging and chemotherapy of tumors and tumor metastasis. (B) UV-vis spectra of


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CuCl2, Pyr-COOH, Pyr-COOH/Cu(II), G5.NHAc/Cu(II), and G5.NHAc-Pyr/Cu(II) in aqueous solution ([Cu] = 0.1 mM for Cu(II) salt and complexes, and Pyr-COOH had equiv. molar concentration to Cu(II)). (C) Digital images of the aqueous solutions of (1) water, (2) Pyr-COOH, (3) CuCl2, (4) Pyr-COOH/Cu(II), and (5) G5.NHAc-Pyr/Cu(II). The Cu concentrations of (3), (4), and (5) were set at 2 mM. (D) Hydrodynamic size distribution of different samples.

For better Cu(II) chelation, Pyr-COOH was partially conjugated on the periphery of G5.NH2 PAMAM dendrimers to afford G5.NH2-Pyr conjugates. Then, the majority of free dendrimer terminal amines were acetylated to give rise to G5.NHAc-Pyr dendrimers, which were characterized by 1H nuclear magnetic resonance (NMR) in Figure S1. Then, the G5.NHAc-Pyr conjugates were used to complex CuCl2 to afford PAMAM dendrimer-Cu(II) nanocomplexes (G5.NHAc-Pyr/Cu(II) complexes). The complexation capacity of G5.NHAc dendrimer and G5.NHAc-Pyr conjugate was evaluated via UVvis spectroscopic titration by addition of CuCl2 (10 equiv. per dendrimer) in aqueous solution at a neutral pH (Figure S2). The number of Cu(II) ions coordinated with each G5.NHAc dendrimer was 50~60, while the number of Cu(II) coordinated with each G5.NHAc-Pyr conjugate was ~60. Due to the presence of 29.7 Pyr moieties per dendrimer, we propose that Cu(II) ions are coordinated to both the Pyr moieties and the dendrimer tertiary amines45 for the G5.NHAc-Pyr conjugates. In the next experiment, we decided to use G5.NHAc-Pyr/Cu(II) and G5.NHAc/Cu(II) complexes coordinating with 50 equiv. Cu(II) per dendrimer in order to avoid the impact of excess Cu(II). As shown in Figure 1B, we compared the UV-vis spectra of CuCl2, Pyr-COOH, Pyr-COOH complexed with CuCl2 (Pyr-COOH/Cu(II)), G5.NHAc dendrimers complexed with 50 molar equiv. of CuCl2 (~108 acetyl groups on the surface of the dendrimer, G5.NHAc/Cu(II) complexes) and G5.NHAc-Pyr/Cu(II) complexes. The absorption peaks of CuCl2 and Pyr-COOH/Cu(II) are at 814 nm and 780 nm, respectively, while those of the G5.NHAc/Cu(II) and G5.NHAc-Pyr/Cu(II) complexes are at 602 and 620 nm, respectively. It seems that the slight change of Cu(II) coordination environment from dendrimer to dendrimer-Pyr leads to the red shift of the absorption peak of Cu(II) complexes. Meanwhile, no absorption peak of Cu(II) (at 814 nm) in the UV-vis spectra for both G5.NHAc/Cu(II) 7 ACS Paragon Plus Environment

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and G5.NHAc-Pyr/Cu(II) complexes were observed, indicating that all the Cu(II) ions were complexed and there were no free Cu(II) ions. Nicely, as depicted in Figure 1C, the intensity of the deep blue color was related to Cu(II) complexation types, following the order of CuCl2 < Pyr-COOH/Cu(II) < G5.NHAc-Pyr/Cu(II). In addition, the G5.NHAc-Pyr/Cu(II) complexes were also characterized by Xray photoelectron spectra (XPS, Figure S3). The XPS spectra of the CuCl2 and G5.NHAc-Pyr/Cu(II) complexes with all binding energies being calibrated by referring to the C 1s (284.9 eV) reveal that Cu 2p region has two different signals of Cu(II) 2p3/2 and Cu(II) 2p1/2 at 934.8 eV and 954.0 eV, respectively for both of them, demonstrating the presence of the Cu(II) in the G5.NHAc-Pyr/Cu(II) complexes. The hydrodynamic sizes and surface potentials of different dendrimers and dendrimeric Cu(II) complexes were measured (Tables S1 and S2). As shown in Figure 1D, after Pyr modification, the G5.NHAc-Pyr conjugates displayed a larger hydrodynamic size (165.9 nm) than the G5.NHAc dendrimers (133.1 nm). The complexation of G5.NHAc-Pyr with Cu(II) gave rise to a decreased hydrodynamic diameter (153.2 nm) of the G5.NHAc-Pyr/Cu(II) complexes when compared to the G5.NHAc-Pyr dendrimers. Similarly, the G5.NHAc/Cu(II) complexes displayed a hydrodynamic diameter of 68.6 nm, which is much smaller than G5.NHAc dendrimers before Cu(II) complexation. Interestingly, these data reveal that there is a tendency to decrease the hydrodynamic size of dendrimers after Cu(II) complexation, which is probably due to the enhanced interaction of Cu(II) with dendrimer backbone to shrink the dendrimer molecular networks. It should be noted that G5 dendrimers display a size of 5.4 nm (http://www.dendritech.com/pamam.html) in diameter, and this size cannot be comparable to that measured by DLS, since DLS measures particles in aqueous solution that may consist many single dendrimer molecules, representing a certain degree of aggregation. In any case, we observed the size and morphology of the G5.NHAc-Pyr/Cu(II) complexes by transmission electron microscopy (TEM), where fuzzy particles with a size around 5.4  0.6 nm for each dendrimer complex 8 ACS Paragon Plus Environment

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can be seen due to the polymeric nature of the complexes. The surface potential of G5.NHAc-Pyr dendrimers was +7.63 mV, in agreement with the literature.46 While after the complexation of Cu(II), the surface potential of G5.NHAc-Pyr/Cu(II) complexes (+8.45 mV) did not change obviously (Figure S4a). The slightly increased zeta potential may be due to the fact that only small part of the copper ions is coordinated to the dendrimer terminal residual primary amines that was resulted from the intrinsic incomplete acetylation.47 After the complexation step, part of the chloride ions is still carried by the particles because some of the Cu(II) have not been fully coordinated by the N and O in the dendrimer backbone. Furthermore, the prepared G5.NHAc-Pyr/Cu(II) complexes also possessed a good colloidal stability. After dispersed in normal saline (NS), cell culture media (both RPMI 1640 medium and DMEM) for at least 2 weeks, no precipitation occurred, suggesting their good stability (Figure S5). We then evaluated the in vitro antiproliferative potency of the dendrimeric Cu(II) complexes using a Cell Counting Kit-8 (CCK-8). As shown in Figure 2A, in the absence of Cu(II), G5.NHAc dendrimers did not have any inhibitory effect against KB cell proliferation in the studied dendrimer concentration range (0.01-10 µΜ). Under similar dendrimer concentrations, G5.NHAc-Pyr conjugates displayed a certain degree of antiproliferative effect and the cell viability decreased to 72.10% at a dendrimer concentration of 10 μM. Clearly, after Cu(II) complexation, the significantly decreased cell viability were noticed by comparing the noncomplexed dendrimers and conjugates (G5.NHAc and G5.NHAcPyr) and their corresponding Cu(II) complexes (G5.NHAc/Cu(II) and G5.NHAc-Pyr/Cu(II)), suggesting that the Cu(II) complexes displayed an obvious inhibition of cell growth. In addition, the G5.NHAcPyr/Cu(II) complexes displayed an increased antiproliferative effect than CuCl2 alone (e.g., 30.19 % versus 53.24 %, at 10 µM), whereas another G5.NHAc/Cu(II) complexes were less cytotoxic than CuCl2 alone (e.g., 57.92 % versus 53.24 %, at 10 µM). Consequently, these results demonstrated that the introduction of Pyr on the surface of PAMAM dendrimers increased the antiproliferative activity, and the Cu(II) complexation further boosted this effect, in agreement with the good antiproliferative 9 ACS Paragon Plus Environment

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activities of G3 phosphorus dendrimers bearing N-(pyridin-2-ylmethylene) ethanamine groups for Cu(II) chelation versus uncomplexed dendrimer reported in the literature.26

Figure 2. (A) The cell viability of KB cells after incubation with G5.NHAc, G5.NHAc-Pyr, G5.NHAc/Cu(II) complexes and G5.NHAc-Pyr/Cu(II) complexes at different dendrimer concentrations for 24 h (mean ± SD, n = 6). (B) IC50 of CuCl2, Pyr-COOH/Cu(II) complexes and G5.NHAc-Pyr/Cu(II) complexes corresponding to various dendrimer concentrations after 24 h incubation with KB cells, A549 cells, C6 cells, and 4T1 cells. For (A) and (B), CuCl2 and Pyr-COOH/Cu(II) had an equiv. molar concentration of Cu(II) in the G5.NHAc-Pyr/Cu(II) complexes. (C) Flow cytometric analysis of 4T1 cells after incubation with CuCl2, Pyr-COOH/Cu(II) complexes, and G5.NHAc-Pyr/Cu(II) complexes at Cu concentration of 1 µM, 10 µM, and 100 µM for 24 h. (D,E) The intracellular ROS level analysis of 4T1 cells after incubation with CuCl2 at Cu concentration of 100 µM, G5.NHAc-Pyr at dendrimer concentration of 2 µM, and G5.NHAc-Pyr/Cu(II) complexes at Cu concentration of 100 µM for 5 h by flow cytometry. (F) Cell cycle analysis of the 4T1 cells after incubation with the G5.NHAc-Pyr/Cu(II) complexes at Cu concentration of 1 µM, 10 µM, 100 µM, and 400 µM, respectively for 24 h. (G) Relative protein expression levels related to S phase and


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apoptosis in 4T1 cells after incubation with CuCl2 or G5.NHAc-Pyr/Cu(II) ([Cu] = 100 µM) for 24 h. The -actin protein was used as a reference for the Western blot assay. (H) Proposed mechanisms underlying the cytotoxicity of G5.NHAc-Pyr/Cu(II) complexes.

The next step was to evaluate the antiproliferative activities of CuCl2, Pyr-COOH/Cu(II) complexes, and G5.NHAc-Pyr/Cu(II) complexes against a panel of various cancer cell lines (Figure 2B). Clearly, the Cu(II)-based dendrimeric complexes have a wide spectrum of anticancer activity. The half maximal inhibitory concentrations (IC50s) of CuCl2, Pyr-COOH/Cu(II) complexes, and G5.NHAc-Pyr/Cu(II) complexes against epidermal carcinoma KB, carcinomic human alveolar basal epithelial A549, glial C6, and murine mammary carcinoma 4T1 cells are shown in Table S3. The Cu(II) complexes of G5.NHAcPyr/Cu(II) and Pyr-COOH/Cu(II) exhibited approximately similar IC50 values against the studied panel of cancer cell lines with IC50s at 4-10 µM. The most sensitive cancer cell line is 4T1 with an IC50 at around 4 µM for both complexes, slightly larger than free Cu(II) salt. Consequently, we focused on 4T1 cells to investigate cytotoxic mechanism of G5.NHAc-Pyr/Cu(II) complexes in the subsequent experiments. Annexin V-FITC (fluorescein isothiocyanate) and PI (propidium iodide) apoptosis detection kit was used to determine the apoptosis effect of 4T1 cells after administration of CuCl2, Pyr-COOH/Cu(II) and G5.NHAc-Pyr/Cu(II). As shown in the density plots (Figure 2C), Cu(II) complexes (Pyr-COOH/Cu(II) and G5.NHAc-Pyr/Cu(II)) markedly increase the proportion of 4T1 cells with phosphatidylserine exposure (Annexin V+) on the outer surface of the plasma membrane and/or loss of plasma membrane integrity (PI+) when compared to the control cells treated with NS. The G5.NHAc-Pyr/Cu(II) complexes display the strongest effect on cell apoptosis among the three materials at all the tested concentrations. The percentage of 4T1 cells at different stages after incubation with the materials for 24 h is presented in Figure S6. Interestingly, the G5.NHAc-Pyr/Cu(II) complexes induce more earlyapoptoptic effect than Pyr-COOH/Cu(II) and CuCl2 at Cu concentration of 1 µM. A significant decrease 11 ACS Paragon Plus Environment

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of early-apoptoptic cell percentage was noticed with G5.NHAc-Pyr/Cu(II) complexes with the Cu concentration of 100 µM, attributing to the transformation of cells from the early stage apoptosis to late stage apoptosis. As expected, the late apoptosis percentage of the G5.NHAc-Pyr/Cu(II) group is significantly higher than those of the Pyr-COOH/Cu(II) and CuCl2 groups for all the studied Cu concentrations (p < 0.01). To explore the underlying mechanisms in the cancer cell apoptosis, the cells treated with the G5.NHAc-Pyr/Cu(II)complexes were subjected to analysis of oxidative stress. The G5.NHAc-Pyr dendrimer conjugates were also tested for comparison. Compared to CuCl2 and G5.NHAc-Pyr, the G5.NHAc-Pyr/Cu(II) complexes induced an obvious intracellular reactive oxygen species (ROS) generation (Figure 2D), which are 2 folds higher than the control group (Figure 2E). The ROS generation data corroborate the apoptosis assay results, suggesting that G5.NHAc-Pyr/Cu(II) complexes are a more potent ROS generator than CuCl2, thereby significantly inducing apoptotic cell death. The higher cellular ROS generation efficacy of the G5.NHAc-Pyr/Cu(II) complexes than that of CuCl2 is likely due to the fact that the dendrimeric Cu(II) complexes display an enhanced intracellular uptake, which has been confirmed by cellular Cu uptake (Figure S7), in agreement with our previous reports.4850

Moreover, the redox status of cells after treated with CuCl2, G5.NHAc-Pyr, and G5.NHAc-Pyr/Cu(II)

complexes for 24 h was tested to determine the different oxidative stress indices, such as glutathione peroxidase (GSH-PX), malondialdehyde (MDA) and superoxide dismutase (SOD). Our data reveal a significant increase in GSH-PX levels for the group of G5.NHAc-Pyr/Cu(II) complexes (Figure S8A). Moreover, compared to control, the SOD activity decreases and the MDA level increases in 4T1 cells after the incubation of G5.NHAc-Pyr/Cu(II) complexes for 24 h (p < 0.05). No significant differences were observed in the groups of CuCl2 and G5.NHAc-Pyr, compared to the control (Figures S8B and 8C). Oxidative stress response of the G5.NHAc-Pyr/Cu(II) complexes was evident by ROS generation, high


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GSH-PX enzyme activity, high lipid peroxidation (which is evident from the increased levels of the MDA), and low SOD enzyme activity. In parallel, the perturbation effect of the G5.NHAc-Pyr/Cu(II) complexes on cell cycle progression was evaluated using flow cytometric detection of DNA content with PI staining technique. The results of the cell cycle distribution analysis for a representative experiment are shown in Figure 2F. Obviously, 4T1 cells after incubated with the G5.NHAc-Pyr/Cu(II) complexes exhibit apparent cell cycle changes. With the increase of Cu concentration for the G5.NHAc-Pyr/Cu(II) complexes, the number of cells with PI-stained DNA content for diploid G2/M period decreases and disappears at Cu concentration of 100 M or above. The percentages of the cellular distribution in different cell cycle phases (G0/G1, S, and G2/M) are given in Figure S9. Compared with the NS-treated control cells, G5.NHAc-Pyr/Cu(II) complexes strongly disturbe the cell cycle. The percentage of cells accumulated in G0/G1 phase and G2/M phase decreases with the increase of Cu concentration. In addition, the percentage of cells in S phase increases continuously with the Cu concentration, indicating that G5.NHAc-Pyr/Cu(II) complexes have induced a S phase arrest. To further clarify the molecular mechanism of the apoptosis and cell cycle arrest caused by the G5.NHAc-Pyr/Cu(II) complexes, the S-phase- and apoptosis-related proteins were determined by Western blotting (Figure 2G). Upon the treatment with the G5.NHAc-Pyr/Cu(II) complexes for 24 h, obvious down-regulation of CyclinA and CDK2 (cyclin-dependent kinase 2) along with up-regulation of CyclinE were observed (Figure S10). That is to say, the G5.NHAc-Pyr/Cu(II) complexes can affect the expressions of cell cycle regulatory proteins thus altering the cell cycle distribution. Additionally, the expression of proapoptotic protein Bax (Bcl-2 Assaciated X protein), suppression of antiapoptotic protein Bcl-2 (B-cell lymphoma-2), tumor suppressor P53 and PTEN (phosphatase and tensin homolog) were also detected (Figure S11). After the cells were treated with CuCl2 or G5.NHAc-Pyr/Cu(II) complexes, the cells display a lower level of Bcl-2, and a higher level of PTEN, Bax and P53 than the 13 ACS Paragon Plus Environment

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control group, which demonstrates the activation of the apoptosis pathway. In addition, the G5.NHAcPyr/Cu(II) group displayed dramatically higher PTEN, Bax and P53 and much more reduction of Bcl-2 than the CuCl2 group (p < 0.001). To summarize, we explored the potential underlying mechanisms related to the therapeutic activity of the G5.NHAc-Pyr/Cu(II) complexes through oxidative stress, cell cycle and apoptosis assays. Our data suggest that the G5.NHAc-Pyr/Cu(II) complexes could alter redox status of cells, resulting in ROS generation and redox homeostasis disruption, followed by cell cycle Sphase arrest and apoptosis, and finally induction of cell death (Figure 2H). Next, to check the MR imaging potential of the G5.NHAc-Pyr/Cu(II) complexes, T1-weighted MR phantom studies were performed. CuCl2 and Pyr-COOH/Cu(II) complexes were also investigated for comparison. Clearly, the G5.NHAc-Pyr/Cu(II)complexes display an Cu concentration-dependent MR signal intensity enhancement trend, which is quite similar to the CuCl2 salt, but much higher than the Pyr-COOH/Cu(II) complexes (Figure 3A). As shown in Figure 3B, the r1 of G5.NHAc-Pyr/Cu(II) complexes was calculated to be 0.7024 mM-1s-1, quite similar to that of free Cu(II) salt (0.7393 mM-1s-1), but around 2 folds higher than that of Pyr-COOH/Cu(II) complexes (0.3572mM-1s-1). The higher r1 value than that of the Pyr-COOH/Cu(II) complexes may be ascribed to the increased globular size of the complexes that renders the Cu(II) to have extended rotational correlation time, similar to the case of Gd(III)-coordinated nanogels reported in our prior work.34 The prepared G5.NHAc-Pyr/Cu(II) complexes were then used for MR imaging of a subcutaneous xenografted 4T1 tumor model. As shown in Figure 3C, the MR signal intensity of the tumor region (as indicated in red circle) increases first and then decreases after the peak time point (70 min) with the time postinjection. The in vivo gray scale T1-weighted MR images of tumor-bearing mice after the tail vein intravenous injection of G5.NHAc-Pyr/Cu(II) complexes are shown in Figure S12. These imaging data suggest that the G5.NHAc-Pyr/Cu(II) complexes are able to accumulate in the tumor region via a passive enhanced permeability and retention (EPR) effect. With the time postinjection, the complexes 14 ACS Paragon Plus Environment

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may have undergone a metabolic process, hence having gradual decrease of Cu(II) accumulation and decreased MR signal intensity. The 4T1 tumor-bearing mice were subjected to in vivo biodistribution analysis at different time points postinjection to evaluate the metabolic pathway (Figure S13). At an earlier time point of 1.5 h, the heart, kidney, and tumor display increased Cu content. After that, the intravenously injected G5.NHAc-Pyr/Cu(II) complexes were taken up predominantly by liver and lung and further be metabolized and gradually cleared from the kidney within 3 days. These results indicate that the dendrimer-based Cu complexes could be accumulated in tumor tissue likely via EPR-based tumor passive targeting pathway and excreted from the body safely, which do not interfere with the copper metabolism of the host animal. For comparison, in the MR images of tumors injected with CuCl2 and Pyr-COOH/Cu(II) complexes (Figure S14), only a slight change of the MR signal intensity was observed during the scanning time range of 0~40 min. This can also be reflected by quantitative analysis of the tumor MR signal-to-noise ratio (SNR) values (Figure S15). It seems that Pyr-COOH/Cu(II) complexes have a slight better MR contrast enhancement than Cu(II) salt, which may be due to the increased molecular weight of the compounds. We also compared the MR contrast enhancement after the tumor-bearing mice were first treated with RT for 3 days (Figure 3D), and the in vivo gray scale MR images are shown in Figure S16. Clearly, there is a noticeable MR contrast enhancement in the tumor region (as indicated in red circle). By quantifying the MR SNR (Figure S17), we can see that the tumor model treated with radiation displays a higher MR SNR than that without radiation treatment at the same time points post intravenous injection of the G5.NHAc-Pyr/Cu(II) complexes. Specifically, the tumor MR SNR increases from 38.84 (preinjection) to 81.66 at 70 min postinjection and remains at 59.29 even at 120 min postinjection, while without radiation, the tumor MR SNR increases from 38.71 (preinjection) to 62.43 at 90 min postinjection and remains at 49.23 at 120 min postinjection. 15 ACS Paragon Plus Environment

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Figure 3. (A) T1-weighted MR images and (B) linear fitting of inverse T1 (1/T1) of CuCl2, G5.NHAc-Pyr/Cu(II) complexes and Pyr-COOH/Cu(II) complexes as a function of Cu concentration (0.05, 0.1, 0.2, 0.4 and 0.8 mM, respectively). The color bar from blue to red indicates the gradual increase of T1 MR signal intensity. The in vivo pseudo-color T1-weighted MR images of mouse with subcutaneous xenografted 4T1 tumor without (C) and with (D) low dose radiation at various time points following the intravenous injection of G5.NHAc-Pyr/Cu(II) complexes. Red circle shows location of tumor.

Taken together, G5.NHAc-Pyr/Cu(II) complexes exhibit more significant MR contrast enhancement and prolonged imaging time when compared to the CuCl2 and Pyr-COOH/Cu(II) under the same conditions. The nanometer size resulting from the high molecular weight of G5.NHAc-Pyr/Cu(II) complexes is believed to be beneficial for their accumulation in the tumor region via the EPR effect. Further, after low dose radiation in the xenografted tumor model, a cascade of changes in the tumor 16 ACS Paragon Plus Environment

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vasculature and microenvironment (such as the vessel enlargement, tortuous vascular branching, and dynamic vascular bursts) combined with enhanced vessel permeability51 render the complexes with enhanced accessibility and penetration ability to the tumor region.52 As a result, the radiation enhances tumor EPR effect to allow the significant tumor accumulation of the G5.NHAc-Pyr/Cu(II) complexes, thereby enabling enhanced tumor MR imaging in vivo. Based on the antiproliferatve effect of G5.NHAc-Pyr/Cu(II) complexes against the 4T1 cell line (vide supra), next we evaluated the potential to use the G5.NHAc-Pyr/Cu(II) complexes as antitumor drug with radiation for RT-enhanced chemotherapy of a subcutaneous xenografted tumor model (the treatment schedule is shown in Figure 4A). As shown in Figure 4B, the body weights of the mice in all groups are negligibly changed, suggesting the low systemic toxicity of G5.NHAc-Pyr/Cu(II) complexes and low dose radiation. Clearly, the tumor growth rate of mice injected with G5.NHAc-Pyr/Cu(II) complexes after a low dose radiation is significantly lower than that of mice treated with NS, NS with radiation, and G5.NHAc-Pyr/Cu(II) complexes. As revealed in Figure 4C, the relative tumor volume after 22 days’ treatment follows the order of G5.NHAc-Pyr/Cu(II) + RT (2.91 ± 0.63 times) < G5.NHAc-Pyr/Cu(II) (4.66 ± 0.59 times) < NS + RT (8.19 ± 1.12 times) < NS (10.56 ± 0.57 times). At the end of these studies, mice were sacrificed, and tumors were collected to measure their weights (Figures 4D and 4E). In comparison with the NS control group, all treated groups show various degrees of inhibition in tumor growth (p < 0.01). Significant differences in the average tumor weight were observed based on the results of the G5.NHAc-Pyr/Cu(II) + RT group when compared with all the other groups (p < 0.01). Then tumor tissue samples were further examined by histological analysis via the hematoxylin and eosin (H&E) staining and the TdT-mediated dUTP Nick-End Labeling (TUNEL) staining. As illustrated in Figure 4F, the tumors in the G5.NHAc-Pyr/Cu(II) + RT group reveal a very high level of necrotic (H&E) and apoptotic (TUNEL) cells, demonstrating the enhanced anticancer effect of the combined RT 17 ACS Paragon Plus Environment

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and chemotherapy. This was also quantitatively confirmed by assessing the cell apoptosis rate, which is the percentage of TUNEL-positive staining area of the apoptotic cells in the tumor sections (Figure S18). The cell apoptosis rate in different groups follows the order of G5.NHAc-Pyr/Cu(II) + RT (87.13 ± 4.01 %) > G5.NHAc-Pyr/Cu(II) (67.45 ± 8.02 %) > NS + RT (43.36 ± 5.16% ) > NS (6.41 ± 1.94%). Similar to the RT-enhanced tumor MR imaging effect, the RT treatment enhances tumor EPR effect that allows for significantly enhanced delivery of the G5.NHAc-Pyr/Cu(II) complexes to the tumor region, thereby enabling improved tumor chemotherapy. Taken together, the above observations suggest that the G5.NHAc-Pyr/Cu(II) complexes exert the in vivo anticancer effects and display the enhanced tumor inhibition ability after low dose radiation.

Figure 4. (A) Schematic illustration of the process of the RT-enhanced chemotherapy using the G5.NHAc-Pyr/Cu(II) complexes. (B) The body weight changes and (C) the relative tumor volumes in 22 days after various treatments (n = 5 in each group). (D) Representative photographs, (E) the average tumor weight, and (F) H&E staining and TUNEL


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staining of the sacrificed tumor tissues on the 22th day post various treatments. The scale bar shown in each panel of (F) represents 100 μm.

To assess the potential in vivo toxicity of G5.NHAc-Pyr/Cu(II) complexes, the major organs such as heart, liver, spleen, lung, and kidney of the surviving mice after treatment with NS, NS + RT, G5.NHAc-Pyr/Cu(II), or G5.NHAc-Pyr/Cu(II) + RT, respectively were sectioned and H&E stained (Figure S19). No noticeable organ damage and appreciable abnormality for all the treatment groups were observed, similar to the NS control. Our results indicate that the designed G5.NHAc-Pyr/Cu(II) complexes combined with low dose radiation potentially do not exert any in vivo systemic toxicity effects in mice after 22 days’ treatment. The G5.NHAc-Pyr/Cu(II) complexes were also used for in vivo MR imaging of lung metastasis, which was compared with a clinical MR contrast agent, Magnevist (Figure 5). The MR contrast enhancement in the lung region in mice bearing lung metastasis after injection of Magnevist is not apparent, whereas that after injection of G5.NHAc-Pyr/Cu(II) complexes is quite obvious (metastatic nodules, as indicated with white arrows). This suggests that the developed G5.NHAc-Pyr/Cu(II) complexes are able to diagnose the 4T1 cells localized in the lung tissues through MR imaging. The MR signal intensity change can be further quantitatively confirmed by plotting the MR SNR as a function of time postinjection. As is illustrated in Figure S20, the MR signal intensity in the lung metastasis nodules after the intravenous injection of G5.NHAc-Pyr/Cu(II) complexes is significantly higher than that after intravenous injection of Magnevist, especially at the time points of 10, 20, and 30 min (p < 0.001). Moreover, histological examinations of the lung tissues were performed using H&E staining assay. The H&E staining of lung tissues in a healthy mouse was used as control (Figure S21). In particular, the increased cell density, metastatic lesions which are denoted as cell clusters with darkly stained nuclei, and neovascularization were noticed by the H&E stained lung tissues in the 4T1 bloodstream metastasis


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model. Overall, the G5.NHAc-Pyr/Cu(II) complexes exhibited more significant MR contrast enhancement to diagnose the lung metastasis when compared to the Magnevist.

Figure 5. T1-weighted grey scale and pseudo-color MR images of mice bearing lung metastasis of 4T1 bloodstream metastasis model at various time points following the intravenous injection of Magnevist (A) and G5.NHAc-Pyr/Cu(II) complexes (B). The white arrow shows location of lung metastatic nodules. (C) Schematic illustration of the process of the RT-enhanced chemotherapy of lung metastasis of a 4T1 bloodstream metastasis model.

The well-established 4T1-breast-cancer lung metastasis model was also utilized to evaluate the therapeutic efficacy of G5.NHAc-Pyr/Cu(II) complexes combined with low dose radiation. The treatment schedule for BALB/c mice with G5.NHAc-Pyr/Cu(II) complexes after low dose radiation is shown in Figure 5C. The RT-enhanced chemotherapy efficacy of the lung metastasis was quantified by 20 ACS Paragon Plus Environment

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calculating the survival rate of mice. As illustrated in Figure S22A, only mice in G5.NHAc-Pyr/Cu(II) + RT group maintain 100% survival rate for a time period of 13 days, and all mice in NS groups as control are dead within 10 days. The survival rate data show that the treatment of G5.NHAc-Pyr/Cu(II) complexes with low dose radiation effectively prolongs the lifetime of lung metastasis mice, further confirming the efficacy of the RT-enhanced chemotherapy. Additionally, the body weights of the mice treated with the G5.NHAc-Pyr/Cu(II) complexes and low dose radiation do not greatly change (Figure S22B). As a result, the considerable tumor inhibition efficacy of the designed G5.NHAc-Pyr/Cu(II) complexes combined with low dose radiation demonstrated a great potential for effective anticancer therapy on the lung metastasis model. It should be noted that in our current study, we only chose Pyr as a ligand to complex Cu(II) for theranostic applications, other ligands53 such as pyrazole, imidazole, 8hydroxyquinoline, etc. may also be linked onto the dendrimer surface to complex Cu(II) for the same purposes. This will be a subject for our on-going studies. In summary, we have designed G5 PAMAM dendrimer-coordinated Cu(II) complexes as a theranostic nanohybrid for T1-weighted MR imaging and RT-enhanced chemotherapy. The Pyrconjugated G5 PAMAM dendrimers (G5.NHAc-Pyr) are able to chelate Cu(II) ions to form stable complexes, representing a simplest design of a nanotheranostic agent having an r1 relaxivity of 0.7024 mM−1s−1. This newly formulated G5.NHAc-Pyr/Cu(II) complexes can upset the redox balance and arrest the cell cycle in S phase, resulting in cell apoptosis. With these properties owned, this novel dendrimer-based Cu(II) complexes can be used for MR imaging of both the xenografted subcutaneous tumor model and the lung metastatic nodules in a bloodstream metastasis model, and enable RTenhanced chemotherapy of both tumor models. Furthermore, the MR imaging performance of G5.NHAc-Pyr/Cu(II) complexes in tumor-bearing mice can be significantly improved after low lose radiation of tumors. With the versatile dendrimer nanotechnology, the developed dendrimer/Cu(II) nanohybrid platform may be further modified with targeting ligands to achieve specific targeting of a 21 ACS Paragon Plus Environment

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particular cancer type for improved cancer theranostics. Overall, our study has strong implications for the rational design of dendrimer-based nanoplatform for better detection and diagnosis in the nanomedicine domain and for translation to clinical trials for RT-adjuvant strategies.

ASSOCIATED CONTENT Supporting Information Detailed materials, methods and additional data of dynamic light scattering, surface potential, IC50 values, 1H NMR, UV-vis titration, XPS survey scan, stability assessment, cell uptake assay, the detection of oxidative stress indices, quantitative analysis of cell apoptosis, cell cycle and Western Blot, in vivo gray scale and pseudo-color T1-weighted MR images, in vivo biodistribution, quantitative analysis of MR SNR, apoptosis rates, H&E staining, and the data of antitumor effect in the lung metastasis of 4T1 bloodstream metastasis model.

AUTHOR INFORMATION Corresponding Authors *E-mail for Xiangyang Shi: [email protected]; phone: + 86-21-67792656 *E-mail for Chen Peng: [email protected]; phone: + 86-21-67792059 *E-mail for Serge Mignani: [email protected]; phone: + 33-688069293 *E-mail for Jean-Pierre Majoral: [email protected]; phone: + 33-561333123 Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT


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This study was financially supported by the National Natural Science Foundation of China (Grant Nos. 81761148028 and 21773026), the Science and Technology Commission of Shanghai Municipality (17540712000), and the Fundamental Research Funds for the Central Universities (CUSF-DH-D2018072).

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Nano Letters

TOC Graphic MR Imaging of Tumor Metastasis

Radiotherapy Enhanced MR Imaging of Tumors

Chemotherapy

Nanotheranostics O NH

O

H N

O

N

N OH N

HN H N

HO N

HN O

NH O

HN

N H O

O

N

O

TUNEL Staining of Tumor Tissues


Poly(amidoamine) Dendrimer-Coordinated Copper(II) Complexes as a

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